In recent years much attention has been focused upon the discovery that biologically active substances can be carried into living cells through the mediation of tiny lipid vesicles, also known as liposomes. Such lipid vesicles comprise monolamellar phospholipid membranes encapsulating aqueous solutions of the biologically active substances and are provided as suspensions in aqueous carriers. The progress of lipid vesicle therapy from the research laboratory to practical medical application has been slow, however, as lipid vesicles are not storage stable and must be prepared immediately before use in order to be fully effective.
A constant supply of dioxygen is required by tissues for the maintenance of metabolism. Many medical situations involve an immediate or prolonged deficiency in the oxygen supply to the tissues, for example, in cardiovascular failure, shock due to blood loss, atherosclerosis and other occlusive vascular disease, erythrocyte enzyme defects, hemolytic anemia, and defects in blood oxygenation due to low oxygen pressure or lung disease. Increase in the supply of oxygen to the red blood cells by administration of gaseous oxygen may have little influence on the amount of oxygen delivered to some tissues. The relatively high oxygen affinity of hemoglobin in red blood cells stored in blood banks and in vivo under certain adverse conditions can result in the release of only a small and inadequate fraction of the hemoglobin-bound oxygen to some critical tissues during circulation.
The normally high oxygen affinity of hemoglobin is reduced in the presence of certain chemical substances known as allosteric effectors, some of which are important natural components of red cells. One of the most potent of these effectors is inositol hexaphosphate, IHP. The incorporation of IHP into intact erythrocytes may be accomplished by incubating the erythrocytes with lipid vesicles containing IHP in a phospholipid membrane, according to the method disclosed by Y-C. Nicolau and K. Gersonde in U.S. Pat. No. 4,192,869, whereby the lipid membrane fuses with erythrocyte cell membrane and the IHP is incorporated into the cell. Upon their return to the circulating blood the IHP-containing erythrocytes release to the tissues a larger fraction of the oxygen they absorbed in the lungs, resulting in increases in the total amount of oxygen supplied to the tissues and allowing a higher tissue oxygen pressure. The reduced oxygen affinity of the hemoglobin contained in the modified erythrocytes is retained for the life of the red cell.
Lipid vesicles laden with IHP begin to lose their ability to cause inositol hexaphosphate to penetrate the erythrocyte membrane within a few hours if they are stored at temperatures above their phase transition temperature. Storage at 37.degree. C. (this temperature is not only the temperature of the physiological environment of the red blood cells but is also higher than the phase transition temperature which is necessary to enhance the fusion of vesicles with the cell membrane) leads to a mutual fusion of the lipid vesicles. This fusion leads to the formation of large multilamellar vesicles which cannot transport the IHP into red cells and thereby decrease their oxygen affinity. FIG. 1 demonstrates the half-life of unilamellar lipid vesicles at 37.degree. C. This half-life time depends on the differences in phospholipid composition. The vesicle-mediated IHP uptake is measured as change in the O.sub.2 half-saturation pressure, of the red blood cells under standard conditions (see also Y. C. Nicolau and K. Gersonde in U.S. Pat. No. 4,192,869).
Until now lipid vesicles could not be stored longer than one to two days and had to be produced anew each time their use was required. The need for the energy- and frequency-controlled sonication procedure at the time and place wherein the modification of the erythrocytes or the administration of lipid vesicle-encapsulated drugs is to be performed limits the usefulness of this procedure and of other medical procedures employing liposome mediated administration of drugs.
The study and potential clinical use of IHP modified erythrocytes is thus severely handicapped by the instability of the lipid vesicles which make it necessary to prepare the IHP-containing lipid vesicles within hours of their use, limiting their use to locations where apparatus is available for preparing the lipid vesicles and increasing the time required for the process of modifying erythrocytes to treat patients. Lipid vesicle suspensions for modifying erythrocytes of patients have to be produced under absolutely sterile conditions. Furthermore, the lipids and the lipid vesicles have to be produced and stored under anaerobic conditions to avoid oxidation of phospholipids and a loss of effectivity to mediate IHP incorporation by cells. These processes therefore have to be performed under servere control, which is possible only in special laboratories. The constraints of lipid membrane instability to heat and oxidation equally limit the usefulness of lipid vesicles laden with biologically active substances other than IHP. The present invention overcomes this problem.